生理学实验室演示：肾小球滤过率在一个老鼠
Summary
The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.
Abstract
Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.
Introduction
The most important function of the kidney is the homeostatic regulation of extracellular water and electrolyte content. The kidneys closely regulate extracellular water, sodium (Na) and potassium (K) to maintain normal physiological levels. Disturbances in renal function can result in serious metabolic disorders which can be fatal. The basic renal process occurs in the nephron and begins with the filtration of plasma at the glomerulus and ends with the excretion of urine. Other processes that determine the final concentration of water, Na and K in the urine are secretion and reabsorption within the nephron. Measurements of glomerular filtration rate (GFR) and the fractional excretion of Na and K are critical in assessing renal function in health and disease. The reader is referred to previously published review articles and textbooks for a more thorough discussion of kidney function1-4.
GFR can be measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron5. While this technique requires anesthesia, surgical preparation, and a terminal experiment, it is considered the gold standard of GFR measurement. Using inulin that is tagged with fluorescein-isothiocyanate (FITC), plasma and urine concentration of FITC-inulin can be easily measured in small volumes and used to calculate GFR during multiple time points of an experiment. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine.
The conceptual understanding of how to measure renal function can easily be demonstrated in a short lab designed to allow students to actively participate in some aspects of the experiment. This video depicts the pre-lab preparation, the renal function demonstration, and the post-lab evaluation of results. The surgical techniques necessary for making measurements of GFR are demonstrated in an anesthetized rat. In addition, example calculations for GFR, and the fractional excretion of Na and K are shown before and after administration of a diuretic drug.
Protocol
Representative Results
Discussion
适当的标记物GFR测量必须符合四个标准：自由过滤在肾小球，可以绑定到血浆蛋白，并没有被吸收，也不分泌的肾单位。菊糖是一种果糖聚合物满足这些标准。其结果是，菊糖的肾清除被认为是金标准测定GFR 7。所表现出的技术代表确定菊粉8,9的恒定输注期间使用定时尿收集菊粉的肾清除率的传统方法。传统菊粉测量已用蒽酮法以产生由分光光度计10,11测得的比色定量测定…
Disclosures
The authors have nothing to disclose.
Acknowledgements
资金来源为实验室示威是NIGMS授予：GM077119。我们感谢博士约瑟夫·海伍德和彼得·科贝特博士为他们的中西医结合和器官系统药理学支持短的：原因。我们也感谢汉娜·加弗女士的技术支持实验室演示。
Materials
| 5-0 Braided Silk Surgical Suture | Surgical Specialties Corp | SP1033 | |
| Assay Plate, 96-Well | Costar | 3922 | |
| Bovine Serum Albumin | Sigma Chemical Co | A2934-25G | |
| Centrifuge | Beckman Coulter | MicroFuge 18, 357160 | |
| Conical Sample Tubes | Dot Scientific Inc. | #711-FTG | |
| Cotton Tipped Applicators | Solon Manufacturing Co | 56200 | |
| Data Acquisition Software | ADInstruments | LabChart Pro 7.0 | |
| Digital Scale | Denver Instrument | APX-4001 | |
| FITC-Inulin | Sigma Chemical Co | F3272-1G | |
| Gauze Sponges | Covidien | 2146 | |
| Heated Surgical Bed | EZ-Anesthesia | EZ-212 | |
| Heparin | Sagnet | NDC 25021-402-10 | |
| HEPES | Sigma Chemical Co | H3375 | |
| Isoflurane | Abbott Animal Health | IsoFlo, 5260-04-05 | |
| Isoflurane Vaporizer | EZ-Anesthesia | EZ-190F | |
| Micro Dissecting Forceps | Biomedical Research Instruments Inc. | 70-1020 | |
| Microplate Reader – Fluoroskan | ThermoScientific | Ascent FL, 5210460 | |
| NOVA 5+ Sodium/Potassium Analyzer | NOVA BioMedical | 14156 | |
| Olsen-Hegar Needle Holders with Scissors | Fine Science Tools | 12002-12 | |
| PE-190 (for bladder catheter) | BD Medical | 427435 | |
| Pressure Transducer | ADInstruments | MLT1199 | |
| Pyrex Culture Tubes | Corning Inc. | 99445-12 | |
| Rat Femoral Tapered Artery Catheter | Strategic Applications Inc. | RFA-01 | |
| Salix Furosemide 5% | Intervet | #34-478 | |
| Strabismus Scissors | Fine Science Tools | 14075-11 | |
| Student Surgical Scissors | Fine Science Tools | 91402-12 | |
| Surgical Gloves | Kimberly-Clark | Sterling Nitrile Gloves | |
| Syringe pump | Razel Scientific | R99-E | |
| Tissue Forceps | Fine Science Tools | 91121-12 | |
| Tissue Scissors | George Tiemann Co | 105-420 |
5-0 Braided Silk Surgical Suture Surgical Specialties Corp SP1033 Assay Plate, 96-Well Costar 3922 Bovine Serum Albumin Sigma Chemical Co A2934-25G Centrifuge Beckman Coulter MicroFuge 18, 357160 Conical Sample Tubes Dot Scientific Inc. #711-FTG Cotton Tipped Applicators Solon Manufacturing Co 56200 Data Acquisition Software ADInstruments LabChart Pro 7.0 Digital Scale Denver Instrument APX-4001 FITC-Inulin Sigma Chemical Co F3272-1G Gauze Sponges Covidien 2146 Heated Surgical Bed EZ-Anesthesia EZ-212 Heparin Sagnet NDC 25021-402-10 HEPES Sigma Chemical Co H3375 Isoflurane Abbott Animal Health IsoFlo, 5260-04-05 Isoflurane Vaporizer EZ-Anesthesia EZ-190F Micro Dissecting Forceps Biomedical Research Instruments Inc. 70-1020 Microplate Reader – Fluoroskan ThermoScientific Ascent FL, 5210460 NOVA 5+ Sodium/Potassium Analyzer NOVA BioMedical 14156 Olsen-Hegar Needle Holders with Scissors Fine Science Tools 12002-12 PE-190 (for bladder catheter) BD Medical 427435 Pressure Transducer ADInstruments MLT1199 Pyrex Culture Tubes Corning Inc. 99445-12 Rat Femoral Tapered Artery Catheter Strategic Applications Inc. RFA-01 Salix Furosemide 5% Intervet #34-478 Strabismus Scissors Fine Science Tools 14075-11 Student Surgical Scissors Fine Science Tools 91402-12 Surgical Gloves Kimberly-Clark Sterling Nitrile Gloves Syringe pump Razel Scientific R99-E Tissue Forceps Fine Science Tools 91121-12 Tissue Scissors George Tiemann Co 105-420
References
- Silverthorn, D. U. . Human Physiology: An integrated approach. , (2012).
- Hall, J. E. . Guyton and Hall Textbook of Medical Physiology. , 303-344 (2011).
- Levey, A. S. Measurement of renal function in chronic renal disease. Kidney International. 38 (1), 167-184 (1990).
- Thurau, K., Valtin, H., Schnermann, J. Kidney. Annual Review of Physiology. 30, 441-524 (1968).
- Shannon, J. A., Smith, H. W. The excretion of inulin, xylose, and urea by normal and phoriziniaed man. Journal of Clinical Investigation. 14, 393-401 (1935).
- Jespersen, B., Knupp, L., Northcott, C. A. Femoral arterial and venous catheterization for blood sampling, drug administration and conscious blood pressure and heart rate measurements. Journal of Visualized Experiments. (59), (2012).
- Sterner, G., et al. Determining ‘true’ glomerular filtration rate in healthy adults using infusion of inulin and comparing it with values obtained using other clearance techniques or predictive equations. Scandinavian Journal of Urology and Nephrology. 42, 278-285 (2008).
- Toto, R. D. Conventional measurement of renal function utilizing serum creatinine, creatinine clearance, inulin and para-aminohippuric acid clearance. Current Opinion in Nephrology and Hypertension. 4 (6), 505-509 (1995).
- Matavelli, L. C., Kadowitz, P. J., Navar, L. G., Majid, D. S. Renal hemodynamic and excretory responses to intra-arterial infusion of peroxynitrite in anesthetized rats. Americam Journal of Physiology. 296, F170-F176 (2009).
- Davidson, W. D., Sackner, M. A. Simplification of the anthrone method for the determination of inulin in clearance studies. Journal of Laboratory, & Clinical Medicine. 62, 351-356 (1963).
- Symes, A. L., Gault, M. H. Assay of inulin in tissues using anthrone. Clinical Biochemistry. 8 (1), 67-70 (1975).
- Shalmi, M., Lunau, H. E., Petersen, J. S., Bak, M., Christensen, S. Suitability of tritiated inulin for determination of glomerular filtration rate. Americam Journal of Physiology. 260 (2 Pt 2), F283-F289 (1991).
- Denton, K. M., Anderson, W. P. Glomerular untrafiltration in rabbits with superficial glomeruli. EUropean Journal of Physiology. 419 (3-4), 235-242 (1991).
- Jobin, J., Bonjour, J. -. P. Measurement of glomerular filtration rate in conscious unrestrained rats with inulin infused by implanted osmotic pumps. Americam Journal of Physiology. 248 (5 Pt 2), F734-F738 (1985).
- Lorenz, J. N., Gruenstein, E. A simple, nonradioactive method for evaluating single-nephron filtration rate using FITC-inulin. Americam Journal of Physiology. 276 (1 Pt 2), F172-F177 (1999).
- Qi, Z., et al. Serial determination of glomerular filtration rate in conscious mice using FITC-inulin clearance. Americam Journal of Physiology. 286 (3), F590-F596 (2004).
- Bivona, B. J., Park, S., Harrison-Bernard, L. M. Glomerular filtration rate determinations in conscious type II diabetic mice. Americam Journal of Physiology. 300 (3), F618-F625 (2011).
- Rosen, S. M. Effects of anaesthesia and surgery on renal hemodynamics. British Journal of Anesthesiology. 44, 252-258 (1972).
- Cousins, M. J. Anesthesia and the kidney. Anaesthesia and intensive care. 11 (4), 292-320 (1983).
- Walter, S. J., Zewde, T., Shirley, D. G. The effect of anaesthesia and standard clearance procedures on renal function in the rat. Quarterly Journal of Experimental Physiology. 74, 805-812 (1989).
- Rieg, T. A. A high-throughput method for measurement of glomerular filtration rate in conscious mice. Journal of Visualized Experiments. (75), (2013).
